Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule

Abstract

Video-enhanced contrast/differential interference-contrast microscopy was used in conjunction with whole mount electron microscopy to study particle transport along linear elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on gold indicator grids and examined with video microscopy. Video records were taken of the linear elements and associated particle transport until lysis and/or fixation of the cells was completed. The preparations were then processed for whole mount electron microscopy. By combining these two methods, we demonstrated that linear elements detected in the living cell could be identified as single microtubules, and that filaments as small as 10 nm could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures changed after lysis with many more cellular components becoming visible. Microtubules became more difficult to detect after lysis while bundles of microfilaments became more prominent. All particle translocations were observed to take place along linear elements composed of one or more microtubules. Furthermore, particles were observed to translocate in one or both directions on the same microtubule.